In VitroCharacterization of Chondrogenic Cells Isolated from Chick Embryonic Muscle Using Peanut Agglutinin Affinity Chromatography

Abstract

Specific binding to the lectin, peanut agglutinin (PNA), has been reported in embryonic precartilage tissues, including the condensing limb bud blastema and the caudal half of the developing somite. The present study aimed to test the hypothesis that PNA-binding may be a surface characteristic of chondroprogenitor cells residing within noncartilage tissues, such as muscle, which have the potential of being induced to form cartilage, e.g., in the presence of bone matrix-derived factors. Day-14 chick embryonic pectoral muscle, which contained histochemically detectable PNA-binding cells, was dissociated into single cells (TM cells) and fractionated by PNA affinity chromatography into PNA-binding (PNA+) and nonbinding (PNA−) cells by PNA-Sepharose 6 MB affinity chromatography. The differentiation potential of the PNA-affinity fractionated cells in vitrowas analyzed as a function of culture plating cell density. Immunohistochemistry of a number of cell-type-specific differentiation markers, including sarcomeric actin, collagen type II, and aggrecan core protein, demonstrated that PNA+ cells, when cultured as a micromass at high density (20 × 10 6cells/ml), exhibited a chondrocyte-like phenotype, whereas the PNA− cells remained myogenic; however, both PNA+ and PNA− monolayer cultures (4 × 10 4cells/ml) behaved as myoblastic cells. The expression of collagen type II mRNA was also confirmed by coupled reverse transcription/polymerase chain reaction analysis. These observations suggest that PNA binding, i.e., the presence of specific galactose-containing cell surface moieties, is likely to be one of the characteristics of chondrogenic cells residing in mesenchymally derived embryonic tissues.